Specific T-cells, which actively suppress unwanted immune responses such as, for example, to endogenous structures (auto-antigens) exist in the organism. They are referred to as regulatory T-cells (Treg). Regulatory T-cells are essentially involved in the life-long maintenance of peripheral tolerance to auto-antigens. They are produced in the thymus, but the activity thereof is greatly reduced with increasing age.
Regulatory T-cells may lead to suppression of anti-tumor responses (Onizuka et al., Cancer Res. 59: 3128-3133 (1999); Shimizu et al., J. Immunol. 163:5211-5218 (1999) both incorporated by reference for all purposes), because many tumor antigens represent classical auto-antigens. By contrast, reduced numbers of Treg or functional alterations in Treg may lead to autoimmune diseases during which endogenous structures are attacked in an uncontrolled manner like foreign substances or pathogens.
Treg are involved in maintenance of immunological self-tolerance, in that they inhibit the activation of auto-reactive T-cells. They are capable of suppressing both cytokine production as well as the proliferation of such potentially pathogenic T-cells. An essential step in the identification of regulatory T-helper cells was the characterization of CD4+ T-helper cells, which include the constitutive alpha-chain protein of the interleukin-2 (IL-2) receptor (CD25) as a surface membrane protein.
The functional significance and the exact molecular mechanisms of the suppression of these CD25+CD4+ regulatory T-cells and also the manner in which they arise have until now not been elucidated. Since the CD25 receptor is also expressed by subpopulations of non-regulatory T-cells, this marker can only be used provisionally for analyzing and concentrating Treg. CD25 cannot, however, be used particularly for identifying and separating activated antigen specific regulatory T-cells.
Isolation of Treg without contaminating conventional T-cells and in particular of Treg specific for certain antigens is, nevertheless, one of the great therapeutic aims. This is also true for the analysis of Treg and antigen-specific Treg. (Auto)antigen-specific Treg are a specific means for suppressing unwanted immune responses such as, for example, autoimmune reactions in rheumatoid arthritis (RA), multiple sclerosis (MS), atherosclerosis (AS), diabetes, and psoriasis. Other unwanted immune responses in which Treg would represent a specific means are GvHD (graft versus host disease) in allogeneic stem cell transplantations or transplant rejection in organ transplantations (Hara et al., J. Immunol. 166: 3789-3796 (2001); Taylor et al., J. Exp. Med. 193: 1311-1318 (2001), both incorporated by reference for all purposes). Allergies also represent unwanted immune responses for which only a few therapeutic options have been available to date. A therapeutic treatment with Treg, which is based on the natural principal of peripheral tolerance and has been demonstrated in many experimental models would involve no side effects by comparison with conventional immunosuppressive medicaments and would be curative.
In relation to the stated importance of Treg, the molecules and mechanisms involved in the suppression, and reliable Treg markers, are of great importance. A molecular biological Treg marker, the transcription repressor FoxP3, which belongs to the Forkhead-family, is acknowledged in the art (Hori et al (2003) Science 99(5609): 1057-61, incorporated by reference for all purposes). In addition, regulatory T-cells can be identified on the bases of expression of the CD25 molecule (Thornton and Shevach (1998) J. Exp. Med. 188 287-296; Sakaguchi et al. (1995) J. Immunol. 155 1151-1164, both incorporated by reference for all purposes).
To date, however, it has not been possible to identify and isolate those Treg cells, which recognize a specific antigen. This would be possible if specifically activated Treg could be separated. This would be a prerequisite for specific therapies with Treg, for example, for autoimmune diseases in which the autoimmune reactions underlying the disease should be suppressed, but not those immune reactions launched against tumor cells.
According to Choi et al. (2004, J. Leukocyte Biology 76:1-7, incorporated by reference for all purposes), no evidence exists that CD137 (4-1BB) can be employed as a discriminative marker for Treg versus CD25. In an international application (WO 2007/110249, incorporated by reference for all purposes), it is described that Treg selectively express CD137 at about 4 hours after activation, whereas conventional T-cells only start to express CD137 after about 12 to 16 hours. Although this early time window can be used to isolate regulatory T-cells only a few hours after activation, this method is prone to errors and therefore is of little practical use. A strong variability in the activation state of the T-cells after obtaining them from blood or tissues as well as the variability of the special kinetics of CD137 on the individual T-cells leads to a contamination of the regulatory T-cells with conventional T-cells, which can be small or large, depending on the time point of the isolation, and which limits the usability of isolated cells dramatically. By using an early time point for separating the cells, the contamination can be kept small, although the yield of the isolated regulatory T-cells is grossly decreased, since not all regulatory T-cells have yet become positive for CD137. As the number of regulatory T-cells, in particular of antigen-specific regulatory T-cells is small to begin with, the decrease in yield diminishes the usability of the method, since many therapeutic uses can no longer be applied.
Therefore, to date, it is not possible to identify and separate the contaminating effector cells (e.g. FoxP3 negative and/or cytokine positive) from the regulatory T-cells (defined by the expression of known markers, in particular CD25, CD25+CD127−, GITR+) or to differentiate antigen-activated regulatory T-cells from activated conventional T-cells.
The term “conventional T cells” as used herein refers to all T cells which do not belong to the “naturally occurring” regulatory T cells which are characterized by stable expression of the transcription factor Foxp3+ and which typically express CD25 constitutively and no or low levels of CD127. Conventional T cells especially refer to T cells which exert immune activating effector functions, i.e. production of effector cytokines such as IL-2, IFN-gamma, IL-4, IL-5, IL-9, IL-17, IL-22.
It is thus difficult, based on the present state of the art, to identify and/or isolate regulatory T-cells. Markers described thus far do not allow the possibility of identifying the totality of regulatory T-cells, because not all regulatory T-cells expressed defined specific markers. Accordingly, it is only possible to identify and/or separate subpopulations, such as, for example only the Treg, which strongly expresses the CD25 receptor. Furthermore, various other cell subpopulations that have the same cell surface markers (such as CD4 for Th1 and Th2, or other Th-cells or CD25 for activated T-cells or B-cells) cannot be distinguished from regulatory T-cells.
In particular, it is not possible to identify activated Treg after stimulation with defined antigens by means of specific activation markers. Although activation of regulatory Th-cells leads to enhanced expression of CD25 and CD38, they are expressed like all other described T-cell activation markers also in other T-cells, so that it has not been possible to separate any antigen-specific Treg by means of specific activation markers.
Frentsch et al. (2005, Nat Med. 11(10):1118-24) and the application EP 1 420 253, both incorporated by reference for all purposes, disclose that CD154 (CD40 ligand) is expressed on all activated T-cells, wherein no distinction is made by the authors between conventional and regulatory T-cells regarding expression of CD154. Therefore, it is not known whether CD154 can be used to discriminate between activated conventional and activated regulatory T-cells.
Rausch et al (2008, Infection and Immunity, 76(5):1908-1919, incorporated by reference for all purposes) disclose that they were able to identify murine antigen-specific Teff and Treg cells by the expression of CD154. The authors in fact assumed that CD154 is also expressed by antigen-activated Treg and could be used for positive selection of antigen-activated Treg.
Therefore, these murine data do not suggest using CD154 as discriminative marker between activated conventional and activated regulatory T-cells.
Most of the methods used to identify and/or isolate regulatory Th-cells on the basis of the expression of a particular cell surface marker depend on recognition of a marker and binding of an antibody. When it is not possible to use a single marker to identify and/or isolate a particular cell type, it is necessary to find a combination of markers and the cognate antibodies (Levings et al., J Exp. Med. 193 (11): 1295-1302 (2001), incorporated by reference for all purposes). Such experiments may in practice be very complicated and difficult to carry out. Moreover, it is possible that the binding of the antibodies influences the activity of the target cell or the expression of other markers, thus having a negative influence on the identification and/or separation process with the other antibodies.
To date, no specific identification and/or separation of exclusively living regulatory T-cells has been carried out. The only specific marker described to date, FoxP3, may moreover, according to recent investigations, be induced also in non-regulatory T-cells (Fontenot J. D. 2003; Hori S. et al. 2003, both incorporated by reference for all purposes). Since FoxP3 is present as an intracellular protein, it is further not possible according to the present state of the art to identify and/or isolate living regulatory cells, for example, on the basis of a FoxP3 antibody. Again, the problem that arises is that only the totality of the Treg can be identified, but not Treg specific for pre-defined antigens. A reliable identification and/or separation of the Treg and in particular of antigen-specific Treg have therefore not been possible.
BD Pharmingen™: “Technical Data Sheet: PE conjugated mouse anti-human CD137 (4-1BB)”, May 7, 2005 (available on the world-wide-web at bdbiosciences.com/external_files/pm/doc/tds/hu-man/live/web_enabled/36005X_555956.pdf, also discloses that the CD137 marker represents an activation marker for all T-cells. The application WO 2005/124346, incorporated by reference for all purposes, also merely describes CD137 as being a marker on CD4+ CD25+; however, the publication does not disclose that CD4+CD25+ especially express at a particular time during activation of the CD137 marker as compared to CD4+ CD25− T-cells. The authors of WO 2005/124346 disclose, on the other hand, that both cell populations of CD4+ CD25+ and CD4+ CD25− T-cells express the CD137 marker to the same extent after activation thereof. The documents mentioned represent the opinion in the art that the CD137 marker cannot be used as a discriminatory marker for CD25+ Treg-cells versus CD25− T-cells. In particular, BD Pharmingen™ describes a PE-conjugated mouse anti-human CD137 antibody. WO 2005/124346 discloses the co-stimulation of freshly isolated CD4+ CD25+ mouse Treg-cells via CD137. The Treg-cells are not stimulated via CD137, but through the presence of strong acting stimuli such as CD3. On the basis of the disclosure mentioned, a skilled artisan assumes that most of the so-called fresh Treg-cells do not express CD137. For example, it is shown in FIG. 5 of WO 2005/124346 that co-stimulation with 4-1BBL exerts activating properties in particular also on CD25− cells. It is disclosed in FIG. 1d in an unambiguous fashion that CD4+ CD25+ and CD4+ CD25− T-cells both express CD137 during activation, so that the publications mentioned do not motivate the skilled artisan to use CD137 for specific presentation or isolation of CD4+ CD25+ Treg-cells from cell mixtures.
In WO 2007/110249, incorporated by reference for all purposes, it is disclosed that Treg express CD137 selectively within an early time frame after activation (only after about 4 hours), whereas conventional T-cells begin to express CD137 after about 12 to 16 hours. Although this time frame may be used for identifying activated regulatory T-cells, the variability of the expression kinetics as well as the short stimulation time leads to a massive decrease of cell purity and yield. There is no method available for enrichment of activated Treg cells allowing the simultaneous depletion of activated conventional T cells and Foxp3+ T cells with instable Foxp3 expression.